Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell line (ab266158)
製品の概要
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製品名
Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell line -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
アプリケーション
適用あり: WBmore details -
Biosafety level
2 -
特記事項
Recommended control: Human wild-type HEK293T cell line (ab255449). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
We will provide viable cells that proliferate on revival.
製品の特性
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Kidney -
Cell type
epithelial -
STR Analysis
Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12 -
Mycoplasma free
Yes -
保存方法
Shipped on Dry Ice. Store in liquid nitrogen. -
バッファー
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
研究分野
ターゲット情報
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関連疾患
Defects in PNP are the cause of purine nucleoside phosphorylase deficiency (PNP deficiency) [MIM:613179]. It leads to a severe T-cell immunodeficiency with neurologic disorder in children. -
配列類似性
Belongs to the PNP/MTAP phosphorylase family. -
細胞内局在
Cytoplasm > cytoskeleton. - Information by UniProt
関連製品
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KO cell lysates
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Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab266158の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 32 kDa.
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特記事項 |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 32 kDa. |
画像
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All lanes : Anti-Nucleoside phosphorylase antibody [EPR5715] (ab109447) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PNP knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 32 kDa
Observed band size: 31 kDa why is the actual band size different from the predicted?Lanes 1-3: Merged signal (red and green). Green - ab109447 observed at 31 kDa. Red - loading control ab7291 observed at 50 kDa.
ab109447 Anti-Nucleoside phosphorylase antibody [EPR5715] was shown to specifically react with Nucleoside phosphorylase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab266158 (knockout cell lysate ab257594) was used. Wild-type and Nucleoside phosphorylase knockout samples were subjected to SDS-PAGE. ab109447 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-Nucleoside phosphorylase antibody [EPR5714] (ab109559) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PNP knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : JAR cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 32 kDa
Observed band size: 31 kDa why is the actual band size different from the predicted?Lanes 1-4: Merged signal (red and green). Green - ab109559 observed at 31 kDa. Red - loading control ab7291 observed at 50 kDa.
ab109559 Anti-Nucleoside phosphorylase antibody [EPR5714] was shown to specifically react with Nucleoside phosphorylase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab266158 (knockout cell lysate ab257594) was used. Wild-type and Nucleoside phosphorylase knockout samples were subjected to SDS-PAGE. ab109559 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Homozygous: 1 bp insertion in exon 2
プロトコール
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (0)
ab266158 は論文での使用が確認できていません。